Specific egg yolk antibody raised to biofilm associated protein (Bap) is protective against murine pneumonia caused by Acinetobacter baumannii

Acinetobacter baumannii easily turns into pan drug-resistant (PDR) with a high mortality rate. No effective commercial antibiotic or approved vaccine is available against drug-resistant strains of this pathogen. Egg yolk immunoglobulin (IgY) could be used as a simple and low-cost biotherapeutic against its infections. This study evaluates the prophylactic potential of IgY against A. baumannii in a murine pneumonia model. White Leghorn hens were immunized with intramuscular injection of the recombinant biofilm-associated protein (Bap) from A. baumannii on days 0, 21, 42, and 63. The reactivity and antibiofilm activity of specific IgYs raised against the Bap was evaluated by indirect ELISA and a microtiter plate assay for biofilm formation. The IgYs against Bap were able to decrease the biofilm formation ability of A. baumannii and protect the mice against the challenge of A. baumannii. IgYs antibody raised here shows a good antigen-specificity and protectivity which can be used in passive immunotherapy against A. baumannii. In conclusion, the IgY against biofilm-associated protein proves prophylactic in a murine pneumonia model.

. The mice were immunized intranasally with chitosan particles harboring the recombinant subunit of Bap (706-1061 region). Passive immunization with sera obtained from these mice conferred 100% protection against 10 × LD 50 of A. baumannii ATCC 19606 in a neutropenic murine pneumonia model.
De Gregorio et al. 17 analyzed Bap in 541 A. baumannii sequenced strains. Their study indicated that Bap was highly polymorphic and was absent among 20% of the sequenced strains. Although Bap is not prevalent in all strains and isolates of A. baumannii 17,18 , it is an antigen of interest to be involved in multi-subunit/multivalent antigens. Hence, it is imperative to introduce novel effective solutions against A. baumannii infections. Recently, specific egg yolk antibodies (IgYs) had been nominated as promising biological macromolecules to be used against A. baumannii pneumonia infection 19,20 . Specific anti-P. aeruginosa IgY successfully advanced to a phase III clinical trial in cystic fibrosis patients (ClinicalTrials.gov Identifier: NCT01455675) 21 . The use of IgYs provides several advantages in comparison to its mammalian counterpart, IgG. Production of IgYs needs no invasive and stressful bleeding of the animal. Egg yolk contains a high amount of IgY which could be purified by simple, costeffective, and environmental-friendly methods 22,23 . It is a safe antibody for passive immunization since it has no interaction with complement and mammalian Fc receptors [23][24][25] . Although it had been revealed that specific IgYs develop protection against A. baumannii, the protection level depends on the antigen of interest 20 . The maximum protection would be expected to be achieved by the combination of the most protective antigens. Hence, further studies to evaluate various antigens are warranted. In the current study, the protectivity of specific anti-Bap IgYs against A. baumannii is assessed in a murine pneumonia model. In this regard, a 371-amino acid region of Bap was recombinantly overexpressed, purified, and injected into laying hens. A clinical isolate of A. baumannii was used to develop a murine pneumonia model. Prophylactic effects of obtained specific antibodies were studied in the developed murine pneumonia model.

Results
Prepared antigen and IgY antibodies. SDS-PAGE analysis showed high purity of the recombinant subunit Bap with a molecular weight of 45 kDa. Western blotting by Anti-His HRP-conjugated antibodies confirmed the recombinant Bap protein (Fig. 1A). SDS-PAGE analysis of purified IgY antibodies revealed their yield and purity (Fig. 1B, C).
Immunoassay results. The immunoreactivity of purified IgY raised against the recombinant protein (Bap) is shown in Fig. 2. The results indicated a significant increasing trend of IgY antibody titer against Bap in immunized hens compared to IgY antibody titer in unimmunized hens (p < 0.0001) ( Fig. 2A, C). Whole-cell ELISA using a clinical strain of A. baumannii as an antigen also confirmed the finding of protein ELISA (p < 0.0001) (Fig. 2B, D). Challenge in a murine pneumonia model. LD 50 was determined as 1.7 × 10 7 CFU. The control IgY-C group receiving only IgY-C antibody (no bacterial challenge), survived for up to 14 days of monitoring. 83% survival was noted in the IgY-Bap group administered with IgY-Bap antibody. All control mice receiving only bacteria died within the first 24 h. All mice receiving IgY-C before the challenge died within 72 h (Fig. 5). Clinical symptoms observed during the 14 days of monitoring included a scruffy coat, squinted eyes, weakness, reduced activity, hunched posture, limping, and finally death (Fig. 6). The mean grade of clinical signs was assessed 20 . Sixteen hours after the inoculation of bacteria, the number of bacteria in the lung and spleen of mice of the IgY-Bap group was at least 2 log 10 CFU/g less than the control group that received only PBS before the challenge (Fig. 7, and Table 1).

Discussion
Acinetobacter baumannii is a gram-negative coccobacillus considered one of the most notorious nosocomial pathogens. Due to the emergence of antibiotic-resistant strains of A. baumannii, the use of non-antibiotic methods such as active and inactive immunization by a vaccine is appreciated. Hence, immunotherapy methods such as the use of polyclonal antibodies (IgY) could be promising against A. baumannii infections. Active immunization of mice with a region of Bap (706-1061 region) could lead to protection in the mice sepsis model against A. baumannii Kh0060 15 . Recently, passive immunization using IgYs raised against the whole cell, OmpA and Omp34 were assessed and showed protection in the pneumonia model. The finding showed an increased survival rate with IgYs against the recombinant protein in comparison to the whole-cell immunization 15,19,20 . Whole-cell ELISA demonstrated that specific IgY antibodies raised against the recombinant subunit Bap could recognize the Bap exposed on the cell surface of A. baumannii. However, as expected, the absorbance of whole-cell ELISA was lower than the purified protein. These findings are consistent with the previous investigations 15, 19,20 . The   26,27 . Inhibition of biofilm formation by antibodies had been reported previously 15 . Notably, the biofilm-disrupting effects of specific anti-Bap IgY were lower than its effect on inhibition of biofilm formation. So, it could be deduced that prophylactic administration of specific anti-Bap IgY is more protective than its therapeutic application. It had In a recent study, mice intranasally immunized with Bap-loaded chitosan particles revealed higher titers of anti-Bap IgG and IgA antibodies in sera as well as lung and fecal samples in comparison to mice immunized with Bap 29 . Titers of IgA in mice subcutaneously immunized with Bap had not been detectable. Passive immunization with sera obtained from mice subcutaneously received Bap had conferred 67% and 34% protection against 5 × LD 50 and 10 × LD 50 respectively. Notably, sera obtained from mice intranasally immunized with Bap-chitosan particles had conferred 100% protection 29 . It seems that the specific anti-Bap IgA could enhance the developed protection against A. baumannii. So, different isotypes of antibodies could confer various protections. The survival rate (83%) through passive immunization by anti-Bap specific IgY in our study, shows higher protectivity in comparison to protectivity conferred by anti-Bap specific IgG 29 . It could be attributed to different strains of A. baumannii using the number of specific antibodies, and/or intrinsic nature and potency of IgY. Further studies need to be conducted to address this issue. Our previous study showed that 20 μg of specific anti-A. baumannii IgYs administered one hour before nasal challenge with viable bacteria could not protect the mice against 10 × LD 50 of A. baumannii ATCC 19606 while 40 μg of these specific IgYs protected the pneumonic mice 19  . Bacterial load per gram spleen or lung tissue 16 h after challenge. The bacterial load was 2 log 10 CFU/g less than the control group, and 1 log 10 CFU/g less than the IgY-C group after 16 h. A significant difference (p < 0.0001) was observed between the IgY-Bap group and control bacteria group as well as the IgY-C group 16 h after the challenge. Such difference was also observed between the IgY-C group and the bacteria group (p < 0.0001). Control: The group without any treatment before infection. IgY-C: the group receiving control IgY antibody 1 h before inoculation with live bacterial suspension. IgY-Bap: the group receiving IgY-Bap antibody 1 h before inoculation. IgY-C: the group receiving only control IgY-C antibody 1 h before inoculation. Control Bacteria group: The group without any treatment before inoculation (****p < 0.0001). www.nature.com/scientificreports/ bacterial population leading to the death of animals in this group after 72 h of challenge. The survival of IgY-Bap group while lungs and liver were loaded with a lower burden of live bacteria in the first 16 h ultimately leading to 83% survival indicates the efficacy of the administered antibodies in the clearance of bacteria from the lungs and liver. This is further supported by the observations of the clinical symptoms where IgY-C group tends to exhibit worsening heath and the IgY-Bap group tends to recover from the infection. Further studies are needed to assess the immune response and decreasing mortality rate by monitoring the bacterial burden in both groups for at least 72 h. The present results, although with limitations, provide evidence for the favorable effect of IgY as a prophylactic and therapeutic modality against A. baumannii. Yet, more research pieces of evidence with animal studies aimed to utilize IgY are vital. Neutrophils are one of the most important factors in the mechanism of action of IgY 30-32 . Since we used neutropenic mice in our study, this mechanism is undermined and the protective effect of IgY could be attributed to the inhibition of biofilm formation and bacterial attachment to the host cell. It would be expected that the protective effect of IgY-Bap be more pronounced in healthy (non-neutropenic) mice.

Groups % Biofilm formation
To the best of our knowledge, this is the first report on the effect of anti-Bap IgY against A. baumannii infections.
The current study could pave the way for the future studies on passive immunization by specific IgYs against the successful nosocomial pathogen, i.e. A. baumannii. In the future studies, various strains of A. baumannii, higher doses of IgY as well as the protective effect of IgY-Bap in non-neutropenic murine pneumonia model could be addressed as limitations of the current study.
The results show that specific anti-Bap IgY could prevent biofilm formation of A. baumannii. Moreover, passive immunization with this IgY could decrease mortality of pneumonic infection caused by A. baumannii in prophylactic mode. Therefore, IgY could be considered promising therapeutics to be entered into clinical phases. Further studies and trials on human subjects could open new perspectives in the application IgY as a therapeutic agent.

Methods
Preparation of Bap subunit. Escherichia coli BL21 (DE3) harboring 706-1076 region of Bap, cloned into pET28a plasmid 15 were grown in LB medium (containing 70 μg/ml kanamycin). Protein expression was performed by the autoinduction method as previously described 33 . The expressed recombinant Bap was purified by using Ni-NTA affinity chromatography (Qiagen) in denaturing conditions and then, was dialyzed against PBS to remove urea. The purity of the obtained recombinant protein was evaluated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The recombinant Bap was confirmed through western blotting with an anti-His tag antibody.
Immunization of hens. The  Preparation and specificity of IgY antibody. The separated egg yolk was diluted (1:7 ratio) with distilled water. PH of the diluted egg yolk was adjusted to 5 with 0.5 M HCl. The mixture was frozen at − 20 °C for 72 h and then was filtered on Whatman cellulose filter paper (Sigma-Aldrich) at room temperature (RT) to remove egg yolk fat. NaCl (8.8%) was added to the clear filtrate and its pH was adjusted to 4. The mixture was stirred for 2 h at RT and then centrifuged at 3700 g for 20 min at 4 °C. The pellet was dissolved in phosphatebuffered saline (PBS, pH 7.4) 34 . The purity and size of IgY were monitored by 10% (w/v) SDS-PAGE. The total concentration of the purified IgY was measured using the Bradford method.

Assessment of specific IgY antibody titers.
Reactivity of specific IgY against whole-cell A. baumannii or the recombinant Bap subunit was assessed by indirect ELISA. Firstly, effect of antigen concentrations (Bap or whole bacterial cell) was evaluated against 10 µg of IgY. In this regard 1.7 × 10 2 -1.7 × 10 8 CFU of a clinical strain of A. baumannii (Kh0060) 15 or 0.0625-10 µg of Bap were coated in a 96 well microplate and incubated overnight at 4 °C. The plate was washed × 3 with 0.5% Tween-PBS (PBST) and was then blocked with 5% skim milk solution in PBST. IgY antibodies at 100 µg/mL concentrations were added to wells. The plate was washed × 3 with PBST after the incubation period was over and was then incubated for one h at 37 °C with 100 μL of 1:1000-diluted HRP-conjugated anti-IgY antibody. The Plate was then washed × 3 with PBST, and TMB substrate (Sigma-Aldrich) was added (100 μL/well; at RT) until the negative control group got colored. Color development was stopped with 2 M H 2 SO 4 , and the absorbance was read at 450 nm. For the main indirect ELI-SAs, 1.7 × 10 7 CFU of A. baumannii (Kh0060) 15 or 5 µg of Bap were coated in a 96 well microplate and incubated overnight at 4 °C. The remaining steps were carried out as described above except for the concentrations of IgY antibodies which were 6.25, 12.5, 25.0, 50.0, and 100 µg/mL.

Biofilm inhibition assay.
Overnight culture of A. baumannii the clinical strain (Kh0060) was inoculated in fresh LB broth medium and allowed to reach OD 600 of 0.6. The bacterial suspension was centrifuged and then pelleted. The precipitate was adjusted to a concentration of 0.5 McFarland standard with PBS and then was added to each well of a 96-well polystyrene microtiter plate with a total volume of 50 µl. BHI, LB, or M9 medium containing 1% glucose and various concentrations (50, 100, and 200 µg/mL) of IgYs were added to each well of the 96-well polystyrene microtiter plate in a total volume of 150 µl. The plates were covered and aerobically incubated at 37 °C for 72 h. Next, the plates were washed twice with PBS (pH 7.4) and stained with 100 μl of www.nature.com/scientificreports/ 0.2% crystal violet (Sigma-Aldrich) for 5 min at RT. The biofilm formation was quantified by measuring the corresponding OD 560 of the supernatant following solubilization with acetone.
Biofilm disruption assay. All steps and desired values were per the previous section (Biofilm inhibition assay) except for the IgYs added after 72 h and the plate was incubated at 37 °C for 2 h. The biofilm formation was quantified by measuring the corresponding OD 560 of the supernatant following solubilization with acetone.
Mice challenge. In mice challenge, four groups (6 mice/group) of female BALB/c mice (22-25 g) were neutropenized by intraperitoneal injection of cyclophosphamide (150 µg per g animal weight) on the first and second days. On the fourth day, all mice were anesthetized by intraperitoneal injection of ketamine (100 µg kg −1 ) and xylazine (20 µg kg −1 ). One hour before the challenge, all groups were intranasally administered with 40 µg IgYs (anti-Bap IgY: IgY-Bap, or control IgY purified from the unimmune hen: IgY-C) or PBS in a total volume of 20 µl. Then, a 10 × LD 50 dose of A. baumannii (Kh0060) 1.7 × 10 8 CFU was administered intranasally in the final volume of 20 µl. One group received only IgY-C antibody purified from the unimmunized hen (Control IgY-C). All groups were monitored for clinical signs for up to 14 days.
Statistical analysis. Statistical analyses were performed by GraphPad Prism 9 (GraphPad Prism version 9.00 for Windows, GraphPad Software, LaJolla, CA, USA). Data were analyzed by one-way analysis of variance with Tukey's multiple comparison test. Survival data for different mice groups were analyzed using the Mantel-Cox log-rank test. Results were expressed as the Mean ± Standard Deviation (SD), and a p value of < 0.05 was considered statistically significant.
Ethics approval. We confirm that this study is reported in accordance with ARRIVE guidelines. The principles stated in the Guide for the Care and Use of Laboratory Animals were followed. The animal care protocol was approved by the ethics committee of Shahed University.

Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.